The rapid development of next-generation sequencing (NGS) technologies has allowed researchers to make unprecedented progress in the analysis of genomes, the results of which have profound implications for human health. The ability to fully characterize genomes remains unrealized, however, due in large part to our inability to reconcile sequence information over long distances. Current long-read, long-template, and synthetic library technologies are underpowered, inconvenient, and expensive. They do not provide a high-throughput, single- platform solution for the de novo assembly of complex genomes. RedVault Biosciences is developing an innovative long template NGS technology that could fundamentally advance our current genome analysis strategies and would have broad applications in the clinic and laboratory. Our previous Phase I study (R43 CA196134) demonstrated feasibility and validated the early components of the technology by creating long template dumbbell (LTD) libraries from genomic DNA and analyzing highly-process, strand-displacing DNA polymerases. Solid-phase rolling circle replication (RCR) was performed from 2-kb and 10-kb genomic LTD libraries and bound products were thoroughly characterized. To demonstrate application using current sequencing technology, we propose a second Phase I grant to show feasibility of our LTD approach with a commercial NGS platform. If successful, our overall goal is to fully develop and translate RedVault's LTD sequencing technology into a robust integrated NGS pipeline capable of resolving complex structural variation and improving de novo genome assembly. Specific Aim 1 will focus on standardizing LTD construction protocols with a commercial, short- read NGS platform. Specific Aim 2 will demonstrate compatibility of RedVault's LTD RCR products with short-read NGS sequencing chemistry using arrayed flow cells, short-read NGS components and reduced complexity single base extension experiments. Specific Aim 3 will optimize compatibility and performance by iteratively sequencing and assembling the C. elegans genome from multiple LTD libraries.